Serotonin (5-HT), a major neurotransmitter, with receptors distributed widely throughout the central and peripheral nervous systems, plays many physiological roles. The majority of serotonin-activated receptors belong to the superfamily of G-protein coupled receptors (GPCRs) (Bradley P B, Engel G, Feniuk W, Fozard J R, Humphrey P P, Middlemiss D N, Mylecharane E J, Richardson B P, Saxena P R (1986) Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology 25:563-576; Hoyer D, Clarke D E, Fozard J R, Hartig P R, Martin G R, Mylecharane E J, Saxena P R, Humphrey P P (1994) International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 46:157-203.; Baez M, Kursar J D, Helton L A, Wainscott D B, Nelson D L (1995) Molecular biology of serotonin receptors. Obes Res 3 Suppl 4:441S-447S; Saxena P R (1995) Serotonin receptors: subtypes, functional responses and therapeutic relevance. Pharmacol Ther 66:339-368; Barnes N M, Sharp T (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38:1083-1152). The 5-HT2A receptor, an important serotonin receptor activates the phospholipase C-IP3 pathway Conn P J, Sanders-Bush E (1984) Selective 5HT-2 antagonists inhibit serotonin stimulated phosphatidylinositol metabolism in cerebral cortex. Neuropharmacology 23:993-996. Pauwels P J (2000) Diverse signalling by 5-hydroxytryptamine (5-HT) receptors. Biochem Pharmacol 60:1743-1750). Agonist-mediated internalization and desensitization of 5-HT2A receptors are dependent on activation of protein kinase C, but desensitization may not directly involve PKC (Berg K A, Stout B D, Maayani S, Clarke W P (2001) Differences in rapid desensitization of 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptor-mediated phospholipase C activation. J Pharmacol Exp Ther 299:593-602; Bhattacharyya S, Puri S, Miledi R, Panicker M M (2002) Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms. Proc Natl Acad Sci U S A 99:14470-14475; Gray J A, Compton-Toth B A, Roth B L (2003) Identification of two serine residues essential for agonist-induced 5-HT2A receptor desensitization. Biochemistry 42:10853-10862.).
Many typical and atypical antipsychotic drugs function as antagonists at 5-HT2A receptors (Canton H, Verriele L, Millan M J (1994) Competitive antagonism of serotonin (5-HT)2C and 5-HT2A receptor-mediated phosphoinositide (PI) turnover by clozapine in the rat: a comparison to other antipsychotics. Neurosci Lett 181:65-68; Meltzer H Y (1999) The role of serotonin in antipsychotic drug action. Neuropsychopharmacology 21 Suppl 1:S106-115; Van Oekelen D, Luyten W H, Leysen J E (2003) 5-HT2A and 5-HT2C receptors and their atypical regulation properties. Life Sci 72:2429-2449). Atypical antipsychotic drugs, in general, have higher affinities for the 5-HT2A receptor than for the dopamine D2 receptor, considered their main target and have limited or no extrapyramidal side effects (Laurier C, Kennedy W, Lachaine J, Gariepy L, Tessier G (1997) Economic evaluation of zuclopenthixol acetate compared with injectable haloperidol in schizophrenic patients with acute psychosis. Clin Ther 19:316-329; Gerlach J (2002) Improving outcome in schizophrenia: the potential importance of EPS and neuroleptic dysphoria. Ann Clin Psychiatry 14:47-57; Marder S R, McQuade R D, Stock E, Kaplita S, Marcus R, Safferman A Z, Saha A, Ali M, Iwamoto T (2003) Aripiprazole in the treatment of schizophrenia: safety and tolerability in short-term, placebo-controlled trials. Schizophr Res 61:123-136). In general assays have concentrated on determining the binding affinities of drugs to 5-HT2A receptors in order to study the possible clinical use of these drugs. Like serotonin, many atypical antipsychotics, viz., clozapine, olanzapine etc. but not typical antipsychotics e.g. haloperidol though antagonists, cause internalization of 5-HT2A receptors both in vitro and a redistribution of the receptor in vivo but the mechanism of internalization and subsequent events remain unknown (Willins D L, Berry S A, Alsayegh L, Backstrom J R, Sanders-Bush E, Friedman L, Roth B L (1999) Clozapine and other 5-hydroxytryptamine-2A receptor antagonists alter the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro and in vivo. Neuroscience 91:599-606). The authors stated that the altered sub-cellular distribution of 5-HT2A receptors within neurons may be related to the therapeutic effects of the atypical antipsychotics. They could not conclude whether the redistribution of the receptor in the dendrites of neurons observed in vivo were the result of internalization or due to a blockage of the transport of newly-synthesized receptors or reduced synthesis . These authors had tested 7 drugs (which would have caused different levels of EPS) and had also not reported any correlation between internalization levels and extrapyramidal symptoms. These experiments had used antibodies against the 5-HT2A receptor to determine internalization.
A typical antipsychotic drugs viz., clozapine, olanzapine etc., show negligible levels of extrapyramidal side effects; drugs such as chlorpromazine show moderate levels of extrapyramidal side effects while typical antipsychotic drugs viz., haloperidol or fluphenazine, cause severe extrapyramidal side effects (Baldessarini, R. J., and Tarazi, F. I. (2001) in Goodman & Gilman's The Pharmacological Basis of Therapeutics (Hardman, J. G., Limbird, L. E., and Goodman Gilman, A., eds), Tenth Ed., pp. 485-520, McGraw-Hill Companies, Inc, USA). Currently there are no available in vitro assays available to screen antipsychotic drugs for extrapyramidal side effects. In vivo assays are essential to determine these effects. It would be therefore useful to develop novel assays that can rapidly provide more on the effect of the drug on the receptor than binding and potentially predict in vivo effects from in vitro assays. Internalization of the receptor, second messenger generation etc. could be suitable but as of now no data exists to provide such predictions.
Internalization of receptors in cell lines have been determined using antibodies directed against various portions of the receptor or by providing fluorescent ligands that induce internalization or by tagging the receptor with a fluorescent protein such as the green fluorescent protein i.e. GFP or a small epitope for which antibodies or ligands are available (Barak L S, Zhang J, Ferguson S S, Laporte S A, Caron M G (1999) Signaling, desensitization, and trafficking of G protein-coupled receptors revealed by green fluorescent protein conjugates. Methods Enzymol 302:153-171). We and others have shown that GFP can be attached to the cytoplasmic tail of the 5-HT2A receptor and the receptor-GFP fusion protein is functional (Bhatnagar A, Willins D L, Gray J A, Woods J, Benovic J L, Roth B L (2001) The dynamin-dependent, arrestin-independent internalization of 5-hydroxytryptamine 2A (5-HT2A) serotonin receptors reveals differential sorting of arrestins and 5-HT2A receptors during endocytosis. J Biol Chem 276:8269-8277; Bhattacharyya S, Puri S, Miledi R, Panicker M M (2002) Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms. Proc Natl Acad Sci U S A 99:14470-14475).
Current pharmacological assays use the binding of and either the activation or inhibition of the receptor to determine the effects of various drugs and internalization has not been suggested as a significant indicator for efficacy of the drug or as a reason for side effects often seen. Interestingly, we and others have observed that a number of antagonists cause internalization (Willins D L, Berry S A, Alsayegh L, Backstrom J R, Sanders-Bush E, Friedman L, Roth B L (1999) Clozapine and other 5-hydroxytryptamine-2A receptor antagonists alter the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro and in vivo. Neuroscience 91:599-60). What we have determined in addition is that for a number of antipsychotics—both typical and atypical their side effects in particular the extrapyramidal symptoms could be directly correlated to its effect on the internalization of the receptor. We also observed that the internalization is also dependent on the cell line used i.e. whether it is a neural or non-neural cell line. Such an assay to classify various antagonists has not been proposed before. We are of the opinion that the effects of drugs on the internalization of the receptor would be a significant and very useful assay to rationally screen for and predict the effectiveness of drugs. Similar assays can also be designed for a large number of receptors that bind to other ligands and should prove extremely useful. Similar receptors can be expressed in non-neuronal and neuronal cells and used to study the internalization of the receptor in these cell lines on application of various ligands. The internalization of the receptor can be determined through fluorescence microscopy. In case of the EGFP the fluorescence can be directly visualized and in case of the Myc-tagged receptor, a monoclonal antibody directed against the Myc epitope is used. Either this antibody or another antibody directed against the Myc antibody is labeled with a fluorophore and used. This assay is used to screen antipsychotics for their ability to cause internalization of the receptor in cells. The ability of the antipsychotic to internalize negatively correlates with the extrapyramidal symptoms the drug causes in vivo.